De Novo Transcriptome Sequencing and Analysis of the Cereal Cyst Nematode, Heterodera avenae Mukesh Kumar 1 , Nagavara Prasad Gantasala 1 , Tanmoy Roychowdhury 2 , Prasoon Kumar Thakur 1 , Prakash Banakar 1 , Rohit N. Shukla 4 , Michael G. K. Jones 3 , Uma Rao 1 * 1 Division of Nematology, Indian Agricultural Research Institute, New Delhi, India, 2 School of Computational & Integrative Sciences, Jawaharlal Nehru University, New Delhi, India, 3 School of Veterinary and Life Sciences, Murdoch University, Perth, Australia, 4 Bionivid Technology [P] Ltd, Bangalore, India Abstract The cereal cyst nematode (CCN, Heterodera avenae) is a major pest of wheat (Triticum spp) that reduces crop yields in many countries. Cyst nematodes are obligate sedentary endoparasites that reproduce by amphimixis. Here, we report the first transcriptome analysis of two stages of H. avenae. After sequencing extracted RNA from pre parasitic infective juvenile and adult stages of the life cycle, 131 million Illumina high quality paired end reads were obtained which generated 27,765 contigs with N50 of 1,028 base pairs, of which 10,452 were annotated. Comparative analyses were undertaken to evaluate H. avenae sequences with those of other plant, animal and free living nematodes to identify differences in expressed genes. There were 4,431 transcripts common to H. avenae and the free living nematode Caenorhabditis elegans, and 9,462 in common with more closely related potato cyst nematode, Globodera pallida. Annotation of H. avenae carbohydrate active enzymes (CAZy) revealed fewer glycoside hydrolases (GHs) but more glycosyl transferases (GTs) and carbohydrate esterases (CEs) when compared to M. incognita. 1,280 transcripts were found to have secretory signature, presence of signal peptide and absence of transmembrane. In a comparison of genes expressed in the pre-parasitic juvenile and feeding female stages, expression levels of 30 genes with high RPKM (reads per base per kilo million) value, were analysed by qRT-PCR which confirmed the observed differences in their levels of expression levels. In addition, we have also developed a user-friendly resource, Heterodera transcriptome database (HATdb) for public access of the data generated in this study. The new data provided on the transcriptome of H. avenae adds to the genetic resources available to study plant parasitic nematodes and provides an opportunity to seek new effectors that are specifically involved in the H. avenae-cereal host interaction. Citation: Kumar M, Gantasala NP, Roychowdhury T, Thakur PK, Banakar P, et al. (2014) De Novo Transcriptome Sequencing and Analysis of the Cereal Cyst Nematode, Heterodera avenae. PLoS ONE 9(5): e96311. doi:10.1371/journal.pone.0096311 Editor: Philippe Castagnone-Sereno, INRA, France Received November 19, 2013; Accepted April 7, 2014; Published May 6, 2014 Copyright: ß 2014 Kumar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors would like to thank the Department of Biotechnology, Government of India for financial support under the Indo-Australia Strategic Research fund (PR12678). M.G.K. Jones thanks the Australia-India Strategic Research Fund (BF030027)for financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: R. Shukla is an employee of Bionivid Technology [P] Ltd. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: [email protected]Introduction The extent of crop losses caused by plant parasitic nematodes is substantial and contributes to significant reductions in crop yields resulting in an annual yield losses of about US$157 billion [1]. The cereal cyst nematode (CCN), Heterodera avenae (Wollenweber, 1924), is one of the three economically important cyst nematode species that attack wheat and barley crops in many cereal growing regions of the world [2]. Although agronomic management practices are often deployed to manage plant nematodes, control with chemical nematicides is also used for high value crops. Because chemical nematicides are toxic and persistent, they are a human health risk and most are being phased out [3]. There is therefore an urgent need to find new gene targets which can be used to develop novel and environmentally friendly nematode control methods [1,4]. Cyst nematodes are obligate sedentary endoparasites that reproduce mainly by amphimixis. After infection of host plants, they develop a close interaction with and feed from a group of interconnected syncytial cells of their host plants. The life cycle starts with an egg present inside an encysted female. The first stage larva (J1) develops within the egg and second stage larva (J2) hatches in response to low soil temperatures (5–15uC) [5]. J2s may invade main or lateral roots in the zone of elongation and migrate intracellularly towards the vascular cylinder [6]. Each J2 selects a single cell that becomes the initial feeding cell (IFC). In a susceptible host, the cells next to the IFC expand and become interconnected after local dissolution of cell walls at pit fields, resulting in the formation of a multinucleate syncytium with characteristics of transfer cells [7]. J2s feed from associated syncytia and undergo three molts, during which they develop either into a mobile male or become a sedentary endoparasitic female that continues to feed until reproduction is completed. The female then dies and forms a cyst which protects the eggs inside it. As a survival strategy not all J2s hatch simultaneously in the same season, but hatching can occur over several years with some unhatched eggs retained within the cyst [8]. Nematodes are amongst the earliest recognised parasites of plants and animals. However, plant parasites have been studied in less detail than many animal parasites and free living nematodes. Most studies have focused on the free living model nematodes, Caenorhabditis elegans and C. briggsae [9] and comparative studies with such nematodes have been useful in annotating genes and PLOS ONE | www.plosone.org 1 May 2014 | Volume 9 | Issue 5 | e96311
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De Novo Transcriptome Sequencing and Analysis of theCereal Cyst Nematode, Heterodera avenaeMukesh Kumar1, Nagavara Prasad Gantasala1, Tanmoy Roychowdhury2, Prasoon Kumar Thakur1,
Prakash Banakar1, Rohit N. Shukla4, Michael G. K. Jones3, Uma Rao1*
1 Division of Nematology, Indian Agricultural Research Institute, New Delhi, India, 2 School of Computational & Integrative Sciences, Jawaharlal Nehru University, New
Delhi, India, 3 School of Veterinary and Life Sciences, Murdoch University, Perth, Australia, 4 Bionivid Technology [P] Ltd, Bangalore, India
Abstract
The cereal cyst nematode (CCN, Heterodera avenae) is a major pest of wheat (Triticum spp) that reduces crop yields in manycountries. Cyst nematodes are obligate sedentary endoparasites that reproduce by amphimixis. Here, we report the firsttranscriptome analysis of two stages of H. avenae. After sequencing extracted RNA from pre parasitic infective juvenile andadult stages of the life cycle, 131 million Illumina high quality paired end reads were obtained which generated 27,765contigs with N50 of 1,028 base pairs, of which 10,452 were annotated. Comparative analyses were undertaken to evaluate H.avenae sequences with those of other plant, animal and free living nematodes to identify differences in expressed genes.There were 4,431 transcripts common to H. avenae and the free living nematode Caenorhabditis elegans, and 9,462 incommon with more closely related potato cyst nematode, Globodera pallida. Annotation of H. avenae carbohydrate activeenzymes (CAZy) revealed fewer glycoside hydrolases (GHs) but more glycosyl transferases (GTs) and carbohydrate esterases(CEs) when compared to M. incognita. 1,280 transcripts were found to have secretory signature, presence of signal peptideand absence of transmembrane. In a comparison of genes expressed in the pre-parasitic juvenile and feeding female stages,expression levels of 30 genes with high RPKM (reads per base per kilo million) value, were analysed by qRT-PCR whichconfirmed the observed differences in their levels of expression levels. In addition, we have also developed a user-friendlyresource, Heterodera transcriptome database (HATdb) for public access of the data generated in this study. The new dataprovided on the transcriptome of H. avenae adds to the genetic resources available to study plant parasitic nematodes andprovides an opportunity to seek new effectors that are specifically involved in the H. avenae-cereal host interaction.
Citation: Kumar M, Gantasala NP, Roychowdhury T, Thakur PK, Banakar P, et al. (2014) De Novo Transcriptome Sequencing and Analysis of the Cereal CystNematode, Heterodera avenae. PLoS ONE 9(5): e96311. doi:10.1371/journal.pone.0096311
Editor: Philippe Castagnone-Sereno, INRA, France
Received November 19, 2013; Accepted April 7, 2014; Published May 6, 2014
Copyright: � 2014 Kumar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors would like to thank the Department of Biotechnology, Government of India for financial support under the Indo-Australia StrategicResearch fund (PR12678). M.G.K. Jones thanks the Australia-India Strategic Research Fund (BF030027)for financial support. The funders had no role in studydesign, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: R. Shukla is an employee of Bionivid Technology [P] Ltd. There are no patents, products in development or marketed products to declare.This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
Figure 1. Distribution of the top 20 species with most homologues to H. avenae. The distribution was calculated using best BLASTX hit.doi:10.1371/journal.pone.0096311.g001
Figure 2. H. avenae orthologues present in selected completely sequenced genomes of free living (C. elegans, Pristionchus pacificus),animal parasitic (A. suum) and plant parasitic (M. incognita, M. hapla, G. pallida, and Pratylenchus thornei) nematodes. The numbersrepresent the common sequences.doi:10.1371/journal.pone.0096311.g002
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Table 3. Comparative analysis of putative RNAi pathway genes in H. avenae, C. elegans and M. incognita.
C. elegans H. avenae M. incognita
Dicer complex
dcr-1 + +
drh-1 + +
drh-3 + +
xpo-1 + +
xpo-2 + +
drsh-1 + +
rde-4 - +
rde-5 - +
RISC complex
alg-1 + +
alg-2 - +
alg-4 + +
ain-1 + +
vig-1 - +
tsn-1 + +
RO6C7.1 - +
CO4F12.1 - +
F58G1.1 - +
T22H9.3 - +
csr-1 + +
sago-1 + -
ppw-1 - -
ppw-2 + -
nrde-3 + -
RdRp amplification complex
ego-1 + +
rrf-1 + +
rrf-3
Systemic RNAi (spreading)
rsd-2 - -
rsd-3 + +
rsd-6 - -
sid-1 - -
sid-2 - -
Required for RNAi
zfp-1 + +
smg-2 + +
smg-6 - -
mes-8 - +
mes-6 - -
mes-2 + +
mut-2 + -
mut-7 + -
gfl-1 + -
cid-1 + +
ekl-4 - +
ekl-1 + +
rha-1 + +
gfl-1 + -
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functional network with 34 nodes and 14 edges using enrichment
map plugin in Cytoscape v 8.1 (Figure 4). Force directed layout
visualisation of the enrichment map showing FDR-based sub
clustering revealed four functional clusters including; a) carbohy-
drate binding b) proteolysis c) motor activity d) hydrolase activity,
hydrolyzing O-glycosyl compounds (Figure 4). Visualisation of
sub-clusters using hierarchical layout suggested that hydrolase
activity was up-regulated in J2s whereas binding activity was up-
regulated in FFs.
Validation of putative differentially expressed genes byqRT-PCR analysis
qRT-PCR was performed to identify some differentially
expressed transcripts by taking cue from transcript quantitation
(Table S2). Thirty genes were selected for further study based on
statistical significance, annotation and presence in at least one
developmental stage (J2 or FF) of H. avenae. Amongst them, 15 genes
were expressed highly in the FF stage viz., (Spectraplakin, Ran-
BPM,CAEBREN_25536, CAEBREN_07082, Receptor family ligand
binding region containing protein, Chitinase-like protein, Superoxide dismutase,
Figure 3. Functional classification of H. avenae transcripts using Gene ontology (GO) terms, a. Biological process, b. Molecularfunction, c. Cellular component.doi:10.1371/journal.pone.0096311.g003
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pectate lyases -PLs (2), carbohydrate esterases - CEs (6) and
carbohydrate binding modules - CBMs (14) (Table S8). A
Figure 4. Hierarchical layout of significantly enriched biological processes and key regulatory genes in H. avenae.doi:10.1371/journal.pone.0096311.g004
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comparative study of the abundance of CAZymes between H.
avenae and M. incognita suggested that M. incognita possessed more
genes for hydrolytic activity. In general, CBMs are the non-
catalytic domains that appends to glycoside hydrolase enzymes
that degrades polysacherides [49–51] (Table S8). Sixty-four CBM
families have been reported in the CAZy database [52] out of
which seven (2, 5, 13, 14, 18, 20 and 50) were found in the H.
avenae transcriptome.
Repeat elements in H. avenae transcripts andidentification of SSRs
The transcriptome data was also used to identify repeat
elements present in H. avenae since there is no information on
genome-wide repeats for this species. The Repeat Masker program
[36] was used to identify different repetitive elements among the
transcripts. Approximately 3% of the total transcripts were found
to be encoded by different repetitive elements (Table S9). Low
complexity regions encoded most transcripts from repetitive
elements. A total of 91 retroelements were found amongst
transcripts, with 43 long interspersed repeat elements (LINEs),
though surprisingly, there were no short interspersed repeat
elements (SINEs). Among retroelements, the number of long
terminal repeats (LTRs) was slightly higher (48) than non-LTR
elements. Also, 28 penelope-like elements distinct from LTR and
non-LTR elements were found [53]. In addition, DNA transpo-
sons of different classes, simple repeats and small RNAs were
identified. For comparison, the C. elegans genome contains about
524 SINE elements of which 46% are encoded in the intronic
regions [54].
Perl script MISA was used to identify SSRs and generated 1,422
SSRs found in 1,125 transcripts, with a frequency of one SSR per
9.33 kb of the sequence (Table S10). Tri-nucleotide SSRs
represented the largest fraction (68.3%) followed by mono-
nucleotide (22.6%) and di-nucleotide (6.6%) repeats. Only a few
tetra- (33) and penta-nucleotide repeat (1) SSRs were identified in
the H. avenae transcripts.
Discussion
In this study, we have sequenced and annotated the tran-
scriptome of two stages of H. avenae after deep sequencing [55].
The combined assembled contigs from RNA-Seq of J2s and FFs
generated 27,765 contigs with N50 of 1,028 bp, for which BLAST
searches yielded 37% (10,454) with significant homologies to
previously annotated genes in standard databases. A comparison
with the EST dataset of NEMABASE4 gave 1,839 clade-specific
unique hits, most from the Heteroderidae that increased transcript
annotation by a further 6%; with 57% of the contigs unchar-
acterized. Comparative analyses of contigs of the J2/FF combined
transcriptome with sequence data from other classes of nematodes
also revealed homologies with free living and animal parasitic
nematodes, and the available H. avenae contigs shared 38.9%
similarity with those of the potato cyst nematode, G. pallida.
Assignment of GO terms categorised 10,751 transcripts to putative
functions.
Our analysis shows that the H. avenae transcriptome encodes
messages most similar to those thought to be involved in parasitism
by G. pallida. To be a successful plant pathogen H. avenae must
locate a host plant root, enter it using its mouth stylet, and migrate
intracellularly from cell-to-cell before inducing the feeding site
syncytium. As has been found for other plant endoparasitic
nematodes, a range of cell wall degrading enzymes have been
identified, possibly required to modify plant cell walls during
migration, feeding or syncytium formation. Thus, the cell wall
modifying CAZymes of H. avenae could be expected to be involved
in these processes. Thirteen GH families were found in H. avenae
transcripts: amongst them, GH5 cellulases are most common to all
the PPNs except for B. xylophilous where GH45 is present. Two b-
1,4 endoglucanases of G. rostochiensis and H. glycines are thought to
be involved in J2 migration to the final feeding site [56]. Some
endoparasitic nematode ‘parasitism’ genes, particularly those
potentially involved in plant cell wall modification, share a high
degree of similarity with genes from bacteria and fungi. This
suggests that horizontal gene transfer (HGT) has occurred during
evolution to parasitism, since such genes are not present in free
living nematodes [56]. The results obtained from H. avenae are
consistent with this view.
Cyst nematodes such as H. avenae must form multinucleate
syncytial feeding sites by altering the differentiation of a group of
cells which fuse together accompanied by local wall degradation.
The nuclei in syncytia become endo-polyploid, and related to this
we have identified 15 transcripts which may be involved in cell
Table 4. Statistically significant regulated Gene Ontology categories and pathways in H. avenae.
Category Term Highly Expressed* Z Score p Value q Value
Cellular component integral to membrane (GO:0016021) 36 1.87 0.23 1.00
Biological process proteolysis (GO:0006508) 11 1.84 0.16 1.00
Molecular function calcium ion binding (GO:0005509) 10 1.68 0.20 1.00
Biological process G-protein coupled receptor protein signaling pathway (GO:0007186) 7 2.15 0.08 1.00
sis, larval development, moulting, growth, reproduction and
defence response to chemicals. An example of such phenotype in
PPNs is the silencing of two genes involved in movement, pat-10
and unc-87, in J2s of P. thornei which reduced reproduction by 77–
81% on carrot mini discs. The phenotypic effect after feeding
dsRNA to these nematodes was abnormal behaviour including
twitching, slow movement, repeated banging of the head against
the body and loss of orientation [72].
Genes involved in the RNAi pathway are well characterised in
many nematode species, although there is less knowledge of the
pathways involved in RNAi in PPNs compared to C. elegans. A total
of 30 genes involved in the RNAi pathway were found amongst
the H. avenae transcripts, including a putative nuclear AGO
(Argonaute) NRDE-3 not previously identified in a PPN (Table 3)
[44] and this needs to be further investigated.
Nematodes have a primitive central nervous system (CNS) and
use signaling processes involving neuropeptides [45] that have a
diverse role in the function and development of nervous system
[73]. They not only act as neuromodulators, but also as primary
transmitters in invertebrate nervous systems [73,74]. To date 109
neuropeptide genes have been identified in C. elegans [73,75,76].
Based on their conserved motifs, these are divided into three
classes: the FMRFamide-like peptide (FLP) gene family, INS genes
that encode insulin-like peptides and peptides derived from
neuropeptide-like protein (NLP) genes which have no sequence
similarity to the other two classes [77]. In the H. avenae
transcriptome data, we found several neuropeptide genes belong-
ing to all the three classes. These included flp-2, 3, 11and 18, four
flp-receptors, nine NLPs and several INS genes such as Insulin-like
growth factor 2. The function of flp gene products is better
understood in C. elegans than in PPNs [44]. Expression studies in C.
elegans using flp-18 reporter gene constructs revealed its expression
in the specific interneurons AVA, AIY and RIG, the motor neuron
RIM and pharyngeal neurons M2 and M3 [78]. flp-18 mutants in
C. elegans were defective in chemosensation, foraging, dauer
formation and fat accumulation and also exhibited decreased
oxygen consumption. flp-18 mutants in C. elegans were defective in
chemosensation, foraging, dauer formation and fat accumulation
and also exhibited decreased oxygen consumption. Host delivered
RNAi of flp-18 significantly reduced the infection and multiplica-
tion of M. incognita [79]. Similar effects could be envisaged from
knock down of these genes in H. avenae.
In addition, to confirm the in silico quantitation, a selection of 30
of the putatively differentially expressed genes were checked by
qRT-PCR and their relative levels of expression validated. High
expression of pectate lyase and b-1, 4 endoglucanase, Annexin and
Figure 5. Confirmation of up regulated genes in the female stage of H. avenae by qRT-PCR. The Y-axis represents the log2 fold changevalues. Error bars show 6SD among the biological replicates. 18S rRNA was used as an internal control gene and fold change was calculated using22DDCT method.doi:10.1371/journal.pone.0096311.g005
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Figure 6. Confirmation of up regulated genes in juvenile stage of H. avenae by qRT-PCR. The Y-axis represents the log2 fold changevalues. Error bars show 6SD among the biological replicates. 18S rRNA was used as an internal control gene and fold change was calculated by using22DDCT method.doi:10.1371/journal.pone.0096311.g006
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host invasion, whereas genes highly expressed in FFs are more
likely to be involved in other metabolic activities such as
reproduction: these results are consistent with similar earlier
observations for cyst nematodes [80]. Up-regulation of CLAVATA
Table 5. Important genes identified in H. avenae based on best BLASTX hits.
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